Intratumor heterogeneity has been demonstrated in a wide range of experimental and human neoplasms. In general, this has been done by isolating tumor subpopulations from single neoplasms and showing them to differ in many characteristics including sensitivity to antineoplastic drugs. We have developed a mouse mammary tumor model system to study the principles of tumor heterogeneity. This system consists of a series of tumor subpopulation lines and sublines, all of which derive ultimately from the same parental tumor. One of the major conclusions of our work is that isolated subpopulations may behave differently when grown alone than they do when they are in each other's presence. Tumor subpopulation interactions confer a societal aspect to cancer growth and behavior. Studies designed to understand the role of tumor heterogeneity in any phenotype need to take the tumor's "ecosystem" into account. In the current grant period we developed an in vitro collagen gel assay to evaluate the sensitivity of tumors to chemotherapeutic drugs. This assay is based on the concepts of tumor heterogeneity including zonal distribution of tumor subpopulations and subpopulation interactions that alter drug response. We are also developing a series of subpopulation lines, companions to the mammary tumor lines of our model system, but which contain specific genetic markers that allow us to quantitate them in mixed populations. Using these lines and our new in vitro assay, we propose to reconstruct heterogeneous tumors by mixing subpopulations that differ in chemosensitivity and in ability to interact with each other and to use the reconstructed tumors to examine the significance of tumor heterogeneity on in vitro and in vivo sensitivity to chemotherapeutic drugs.